Members of the order Kinetoplastida, including the causative agents of African sleeping sickness (Trypanosoma brucei subspecies), leishmaniasis (Leishmania spp.), and Chagas disease (Trypanosoma cruzi) possess a unique subcellular organelle, the glycosome. This organelle houses many of the enzymes of the Embden-Meyerhof pathway of glycolysis, as wells as enzymes involved in nucleotide biosynthesis, ether-lipid biosynthesis, and carbon dioxide fixation. There is no vaccine against any of these diseases and chemotherapy is toxic. A further understanding of the glycosomal organelle might lead to the development of new chemotherapies, particularly in the case of T. brucei where the parasites rely completely on glycosomal metabolism for the generation of energy while in the mammalian host. Our studies are aimed at analyzing specific constituents of the glycosome and the glycosomal biogenesis system, to further elucidate unique aspects of glycosomal metabolism and its role in the survival of the organism. Using transient transfection and a luciferase reporter system, experiments will identify and analyze the signals on proteins which target them to the glycosome. Gene knockout and gene replacement studies will examine the role of a phosphoglycerate kinase found only in the glycosome of Kinetoplastida. Glycosome-deficient mutants of T. brucei and Leishmania will be generated and studied to determine if the lack of the glycosome attenuates the parasite. These studies will clarify the role of the glycosome in Leishmania, which do not rely as extensively on glycolysis as does T. brucei. In addition, the mutants will also serve as the starting point for genetic experiments aimed at isolating genes important in glycosomal biogenesis. Insight into these crucial parameters of glycosomal biogenesis may identify unique aspects which may be selectively disrupted, and will allow us to determine whether the glycosome is an appropriate target for chemotherapy of diseases caused by trypanosomes and Leishmania.